Creatine amidinohydrolase, production thereof and use thereof

ABSTRACT

A creatine amidinohydrolase having the following physicochemical properties:
         Action: catalyzing the following reaction;
           creatine+H 2 O→sarcosine+urea   
           Optimum temperature: about 40-50° C.   Optimum pH: pH about 8.0-9.0   Heat stability: not more than 50° C. (pH 7.5, 30 min)   Km value for creatine in a coupling assay using a sarcosine oxidase and a peroxidase: about 3.5-10.00 mM   Molecular weight: about 43,000 (SDS-PAGE)   Isoelectric point: 3.5 4.5,
 
a method for producing said enzyme, comprising culture of microorganism producing said enzyme, a method for the determination of creatine or creatinine in a sample using said enzyme, and a reagent therefor.

CROSS-REFERENCE TO RELATED APPLICATIONS

More than one reissue application has been filed for the reissue of U.S.Pat. No. 6,080,553. The reissue applications are the present applicationand application Ser. No. 09/940,941, each of which is a divisionalreissue of U.S. Pat. No. 6,080,553.

FIELD OF THE INVENTION

The present invention relates to a novel creatine amidinohydrolase,specifically, a novel creatine amidinohydrolase having a very low Kmvalue for creatine, and a method for producing said enzyme. The presentinvention also relates to a method for the determination of creatine orcreatinine in a sample by the use of said enzyme, and a reagenttherefor.

BACKGROUND OF THE INVENTION

A creatine and a creatinine are found in blood and urine. A quick andaccurate determination of their amounts is very important in makingdiagnosis of the diseases such as uremia, chronic nephritis, acutenephritis, giantism, tonic muscular dystrophy and the like. For makingdiagnosis of these diseases, creatine and creatinine in blood, as wellas urine are frequently determined quantitatively.

A creatine can be determined by allowing creatine aminohydrolase andsarcosine oxidase to react on creatine in a sample and determining theamount of the generated hydrogen peroxide by a method for measuringhydrogen peroxide. A creatinine can be determined by allowing creatinineamidohydrolase, creatine amidinohydrolase and sarcosine oxidase to reacton creatinine in a sample and determining the generated hydrogenperoxide by a method for measuring hydrogen peroxide.

The creatinine amidohydrolase, creatine amidinohydrolase and sarcosineoxidase are widely found in the world of microorganisms, have beenindustrially produced and used as reagents for clinical tests.

Yet, the creatine amidinohydrolase produced from various known celllines show lower heat stability and greater Km value for creatine. Forexample, an enzyme derived from the bacteria belonging to the genusBacillus (U.S. Pat. No. 4,420,562) is thermally stable only at atemperature not more than 40° C. An enzyme derived from Pseudomonasputida has a smaller apparent Km value for creatine of 1.33 mM [ArchivesBiochemistry and Biophysics 177, 508-515 (1976)], though the method fordetermining the activity is different and the Km value for creatinedetermined by a coupling assay using sarcosine oxidase and peroxidasewidely used as reagents for clinical tests, has been unknown. Theenzymes derived from the bacteria belonging to the genusCorynebacterium, Micrococcus, Actinobacillus or Bacillus (JapanesePatent Examined Publication No. 76915/1991) is thermally stable at atemperature not more than 50° C., whereas Km value for creatine is asgreat as about 20 mM, and these enzymes are not suitable for use asreagents for clinical tests.

In an attempt to resolve such problems, the present inventors previouslyfound that the bacteria belonging to the genus Alcaligenes produced acreatine amidinohydrolase which was superior in heat stability and had arelatively smaller Km value (Km value: ca. 15.2) for creatine (JapanesePatent Unexamined Publication No. 63363/1994). Furthermore, they haveestablished a technique for isolating a creatine amidinohydrolase genehaving a relatively small Km value for creatine from said bacterial cellline and producing said enzyme in a large amount using Gram negativebacteria as a host (Japanese Patent Application No. 117283/1995).

Moreover, a creatine amidinohydrolase stable in a high pH range andhaving a small Km value has been reported to be derived from the samegenus Alcaligenes cell line (U.S. Pat. No. 5,451,520).

Yet, these creatine amidinohydrolases still have greater Km values asenzymes to be used as routine reagents for clinical tests, and acreatine amidinohydrolase having smaller Km value has been desired.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to produce a novelcreatine amidinohydrolase having a small Km value for creatine to theextent sufficient for use as a general reagent for clinical tests,preferably not more than about 15.0, and provide a means for determiningcreatine or creatinine in a sample using said enzyme.

The present invention is based on the successful provision of a creatineamidinohydrolase gene which expresses a novel creatine amidinohydrolasehaving a small Km value for creatine, by introducing a mutation, bygenetic engineering and protein engineering, into a creatineamidinohydrolase gene derived from conventionally known bacteriabelonging to the genus Alcaligenes, which is a known creatineamidinohydrolase having a rather small Km value. The creatineamidinohydrolase of the present invention can be produced in largeamounts by culturing a microorganism capable of expressing said gene ina nutrient medium.

The novel creatine amidinohydrolase of the present invention has a verysmall Km value for creatine as compared to conventionally known enzymes,and shows superior reactivity to creatine contained in a trace amount ina sample. Thus, it is useful as a reagent for determining creatine orcreatinine with high sensitivity and high precision.

Accordingly, the present invention provides a novel creatineamidinohydrolase having the following physicochemical properties.

-   -   Action: catalyzing the following reaction:        -   creatine+H₂O→sarcosine+urea    -   Optimum temperature: ca. 40-50° C.    -   Optimum pH: ca. 8.0-9.0    -   Heat stability: stable at not more than about 50° C. (pH 7.5, 30        min)    -   Km value relative to creatine in a coupling assay using a        sarcosine oxidase and a peroxidase: ca. 3.5-10.0 mM    -   Molecular weight: ca. 43,000 (SDS-PAGE)    -   Isoelectric point: ca. 3.5 4.5

The present invention also provides a method for producing said creatineamidinohydrolase, comprising culturing a microorganism capable ofproducing a novel creatine amidinohydrolase having the followingphysicochemical properties, in a nutrient medium, and harvesting saidcreatine amidinohydrolase from the culture.

-   -   Action: catalyzing the following reaction:        -   creatine+H₂O→sarcosine+urea    -   Optimum temperature: ca. 40-50° C.    -   Optimum pH: ca. 8.0-9.0    -   Heat stability: stable at not more than about 50° C. (pH 7.5, 30        min)    -   Km value relative to creatine in a coupling assay using a        sarcosine oxidase and a peroxidase: ca. 3.5-10.0 mM    -   Molecular weight: ca. 43,000 (SDS-PAGE)    -   Isoelectric point: ca. 3.5 4.5

The present invention further provides a reagent for determiningcreatine in a sample, comprising the above-said creatineamidinohydrolase, sarcosine oxidase and a composition for detection ofhydrogen peroxide, and a method for determining creatine in a sample bythe use of said reagent.

The present invention further provides a reagent for determiningcreatinine in a sample, comprising a creatinine amidohydrolase, theabove-mentioned creatine amidinohydrolase, sarcosine oxidase and acomposition for detection of hydrogen peroxide, and a method fordetermining creatinine in a sample by the use of said reagent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a physical map of recombinant plasmid pCRH273.

FIG. 2 shows the time course determination results of creatinine in asample, by the use of the creatine amidinohydrolase of the presentinvention and a wild creatine amidinohydrolase.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the present invention is a novel creatineamidinohydrolase having the following physicochemical properties.

-   -   Action: catalyzing the following reaction:        -   creatine+H₂O→sarcosine+urea    -   Optimum temperature: ca. 40-50° C.    -   Optimum pH: ca. 8.0-9.0    -   Heat stability: stable at not more than about 50° C. (pH 7.5, 30        min)    -   Km value relative to creatine in a coupling assay using a        sarcosine oxidase and a peroxidase: ca. 4.5±1.0 mM    -   Molecular weight: ca. 43,000 (SDS-PAGE)    -   Isoelectric point: ca. 3.5 4.5

Another embodiment of the present invention is a novel creatineamidinohydrolase having the following physicochemical properties.

-   -   Action: catalyzing the following reaction:        -   creatine+H₂O→sarcosine+urea    -   Optimum temperature: ca. 40-50° C.    -   Optimum pH: ca. 8.0-9.0    -   Heat stability: stable at not more than about 50° C. (pH 7.5, 30        min)    -   Km value relative to creatine in a coupling assay using a        sarcosine oxidase and a peroxidase: ca. 6.5±1.0 mM    -   Molecular weight: ca. 43,000 (SDS-PAGE)    -   Isoelectric point: ca. 3.5 4.5

A still another embodiment of the present invention is a novel creatineamidinohydrolase having the following physicochemical properties.

-   -   Action: catalyzing the following reaction:        -   creatine+H₂O→sarcosine+urea    -   Optimum temperature: ca. 40-50° C.    -   Optimum pH: ca. 8.0-9.0    -   Heat stability: stable at not more than about 50° C. (pH 7.5, 30        min)    -   Km value relative to creatine in a coupling assay using a        sarcosine oxidase and a peroxidase: ca. 9.0±1.0 mM    -   Molecular weight: ca. 43,000 (SDS-PAGE)    -   Isoelectric point: ca. 3.5 4.5

One method for producing the creatine amidinohydrolase of the presentinvention comprises mutation of a gene encoding a wild creatineamidinohydrolase by genetic engineering and protein engineering method,generating a mutant DNA encoding a novel creatine amidinohydrolasehaving a smaller Km value for creatine than the wild creatineamidinohydrolase, expressing said DNA in a suitable host and harvestingthe creatine amidinohydrolase thus produced.

While the gene encoding a wild creatine amidinohydrolase which is to bemutated is not particularly limited, in one embodiment of the presentinvention, it is the creatine amidinohydrolase gene depicted in theSequence Listing•SEQ ID:No.2, which is derived from Alcaligenes•faecalisTE3581 (FERM P-14237).

In another embodiment of the present invention, a novel creatineamidinohydrolase having a smaller Km value for creatine than a wildcreatine amidinohydrolase is produced by mutating the gene encoding theamino acid sequence depicted in the Sequence Listing•SEQ ID No:1.

A wild creatine amidinohydrolase gene can be mutated by any knownmethod. For example, a wild creatine amidinohydrolase DNA or amicroorganism cells having said gene is brought into contact with amutagenic agent, or ultraviolet irradiation is applied, or a proteinengineering method is used such as PCR and site-directed mutagenesis.Alternatively, an Escherichia coli susceptible to gene mutation at highfrequency due to defective gene repair mechanism may be transformed witha wild creatine amidinohydrolase gene DNA for mutation in vivo.

For example, Escherichia coli is transformed with the mutant creatineamidinohydrolase gene obtained above and plated on a creatineamidinohydrolase activity detection agar medium [J. Ferment. Bioeng.,Vol. 76 No. 2 77-81(1993)], and the colonies showing clear colordevelopment are selected. The selected colonies are inoculated to anutritive medium (e.g., LB medium and 2×YT medium) and culturedovernight at 37° C. The cells are disrupted and a crude enzyme solutionis extracted.

The method for disrupting the cells may be any known method, such asphysical rupture (e.g., ultrasonication and glass bead rupture), as wellas by the use of a lysozyme. This crude enzyme solution is used todetermine the creatine amidinohydrolase activity of two kinds ofactivity determination reaction solutions having different substrateconcentrations. Comparison of the activity ratios of the two with thatobtained using a wild creatine amidinohydrolase leads to the screeningof the creatine amidinohydrolase having smaller Km value.

The method for obtaining the purified creatine amidinohydrolase from thecell line selected as above may be any known method, such as thefollowing.

After the cells obtained by culturing in a nutrient medium arerecovered, they are ruptured by an enzymatic or physical method andextracted to give a crude enzyme solution. A creatine amidinohydrolasefraction is recovered from the obtained crude enzyme solution byammonium sulfate precipitation. The enzyme solution is subjected todesalting by Sephadex G-25 (Pharmacia Biotech) gel filtration and thelike.

After this operation, the resulting enzyme solution is separated andpurified by octyl Sepharose CL-6B (Pharmacia Biotech) columnchromatography to give a standard purified enzyme product. This productis purified to the degree that it shows almost a single band bySDS-PAGE.

The microorganism to be used in the present invention to produce thenovel creatine amidinohydrolase is exemplified by Escherichia coli JM109(pCRH273M1) (FERM BP-5374), Escherichia coli JM109 (pCRH273M2) (FERMBP-5375), Escherichia coli JM109 (pCRH273M3) (FERM BP-5376) and thelike.

The method for culturing these microorganisms and recovering thecreatine amidinohydrolase of the present invention from the culturesthereof are not particularly limited, and conventional methods can beapplied.

The novel creatine amidinohydrolase obtained by the above-mentionedproduction method of the present invention has the followingphysicochemical properties.

-   -   Action: catalyzing the following reaction:        -   creatine+H₂O→sarcosine+urea    -   Optimum temperature: ca. 40-50° C.    -   Optimum pH: ca. 8.0-9.0    -   Heat stability: stable at not more than about 50° C. (pH 7.5, 30        min)    -   Km value relative to creatine in a coupling assay using a        sarcosine oxidase and a peroxidase: ca. 3.5-10.0 mM    -   Molecular weight: ca. 43,000 (SDS-PAGE)    -   Isoelectric point: ca. 3.5 4.5

The Km value in the present invention is the value relative to creatinein a coupling assay using a sarcosine oxidase and a peroxidase. Whilethe conventional enzyme derived from Pseudomonas putida has a smallapparent Km value for creatine of 1.33 mM [Archives Biochemistry andBiophysics 177, 508-515 (1976)], the activity is determined by measuringthe residual creatine in the reaction mixture with α-naphthol anddiacetyl, and the Km value for creatine by a coupling assay using asarcosine oxidase and a peroxidase, which are widely used as reagentsfor clinical tests, has been unknown.

The creatine amidinohydrolase of the present invention can be used forthe determination of creatine upon combination with a sarcosine oxidaseand a composition for detection of hydrogen peroxide. Moreover, whencreatinine amidohydrolase is concurrently used, creatinine can bedetermined as well.

The determination method of the present invention utilizes the followingreactions.

When creatinine is determined, the following reaction is furtherutilized.

The quinonimine pigment produced is generally subjected to thedetermination of absorbance at 500-650 nm wavelength. The method fordetermining creatine is an end method or a rate method, though the endmethod is generally used.

The inventive creatine amidinohydrolase having smaller Km value canreduce the amount of the enzyme to be used in the test reagent forcreatine or creatinine determination to about ⅓-¼ as compared to thenecessary amount of conventional enzymes, and achieves good reactivityin the latter half of the reaction.

The reagent for determining creatine in a sample of the presentinvention contains the above-mentioned creatine amidinohydrolase,sarcosine oxidase, and a composition for detecting hydrogen peroxide.

The reagent for determining creatinine in a sample of the presentinvention contains a creatinine amidohydrolase, the above-mentionedcreatinine amidohydrolase, sarcosine oxidase, and a composition fordetecting hydrogen peroxide.

The sarcosine oxidase to be used for detecting creatine or creatinine ofthe present invention can be obtained from the microorganisms originatedfrom the genera Arthrobacter, Corynebacterium, Alcaligenes, Pseudomonas,Micrococcus, Bacillus and the like, and some of them are commerciallyavailable.

The creatinine amidohydrolase can be obtained from the microorganismsoriginated from the genera Pseudomonas, Flavobacterium, Alcaligenes,Penicillium and the like, and some of them are commercially available.

The composition for the detection of hydrogen peroxide contains anenzyme having a peroxidase activity, chromophore and a buffer. Theenzyme having a peroxidase activity is exemplified by peroxidase,haloperoxidase, bromoperoxidase, lactoperoxidase, myeloperoxidase andthe like. The chromophore comprises a hydrogen receptor and a coupler.The hydrogen receptor may be any as long as it receives hydrogen in thereaction with hydrogen peroxide, peroxidase and a coupler, which isspecifically exemplified by 4-aminoantipyrine,3-methyl-2-benzothiazoline-hydrazine derivative and the like. Examplesof the coupler include aniline derivatives such as aniline andN-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (TOOS), phenolderivatives such as phenol and p-chlorophenol, and the like.

The reagent for the determination of creatine of the present inventioncontains each ingredient in a preferable proportion of creatineamidinohydrolase ca. 5-300 U/ml, sarcosine oxidase ca. 1-100 U/ml,peroxidase ca. 0.01-50 U/ml, hydrogen donor ca. 0.1-10 mM, and a couplerca. 0.1-50 mM.

The reagent for the determination of creatinine of the present inventioncontains each ingredient in a preferable proportion of creatinineamidohydrolase ca. 10-300 U/ml, creatine amidinohydrolase ca. 10-300U/ml, sarcosine oxidase ca. 1-100 U/ml, peroxidase ca. 0.01-50 U/ml,hydrogen donor ca. 0.1-10 mM, and a coupler ca. 0.1-50 mM.

The reagent for the determination of creatine or creatinine of thepresent invention is generally used with a buffer having a pH of about6-8. Examples of the buffer include phosphate buffer, Good buffer, Trisbuffer and the like.

Where necessary, ascorbate oxidase or catalase may be added to thereagent of the present invention. Other compounds may be also added tothe reagent of the present invention for smooth enzyme reaction andcolor development. Such compounds are, for example, stabilizers,surfactants, excipients and the like.

EXAMPLES

The present invention is described in detail by way of the followingExamples.

In the Examples, the activity of creatine amidinohydrolase wasdetermined as follows. The enzyme activity in the present invention isdefined to be the enzyme amount capable of producing 1 μmole ofsarcosine per min under the following conditions being one unit (U).

Reaction mixture composition 0.3 H HEPES pH 7.6 0.005% 4-aminoantipyrins0.015% phenol 1.8% creatine 6 U/ml sarcosine oxidase 6 U/ml perioxidase

The above-mentioned reaction mixture (3 ml) is taken with a cuvette (d=1cm) and preliminarily heated to 37° C. for about 3 minutes. An enzymesolution (0.1 ml) is added, and the mixture is gently admixed. Usingwater as a control, changes in absorbance at 500 nm are recorded for 5minutes using a spectrophotometer controlled to 37° C. Based on thelinear portion of 2-5 minutes thereof, changes in absorbance per minuteare determined (ΔOD) test.

The blank test is performed in the same manner as above except that asolution (0.1 ml, 50 mM potassium phosphate buffer, pH 7.5) for dilutingthe enzyme is used instead of the enzyme solution and changes inabsorbance per minute are determined (ΔOD blank).

The enzyme amount is calculated by inserting each measure into thefollowing formula. $\text{U/ml} = \frac{\begin{matrix}{\text{ΔOD/min~~(ΔOD~~text} -} \\{\text{ΔOD~~blank)} \times \text{3.1} \times \text{dilution~~fold}}\end{matrix}}{13.3 \times {1/2} \times 1.0 \times 0.1}$wherein each constant denotes the following:

-   -   13.3: millimolar absorbance coefficient (cm²/μM) under the above        measurement conditions of quinonimine pigment    -   ½: coefficient indicating that the quinonimine pigment formed        from one molecule of hydrogen peroxide generated in the enzyme        reaction is ½ molecule    -   1.0: light path length (cm)    -   0.1: amount of enzyme added (ml)

Reference Example 1

Isolation of chromosomal DNA

The chromosomal DNA of Alcaligenes•faecalis TE3581 was isolated by thefollowing method.

The cells (FERM P-14237) were shake-cultured overnight at 30° C. in anutrient broth (150 ml) and the cells were collected by centrifugation(8000 rpm, 10 min). The cells were suspended in a solution (5 ml)containing 10% sucrose, 50 mM Tris-HCl (pH 8.0) and 50 mM EDTA, and alysozyme solution (1 ml, 10 mg/ml) was added. The mixture was incubatedat 37° C. for 15 min. Then, 10% SDS solution (1 ml) was added. Anequivalent amount (1 ml) of a chloroform•phenol solution (1:1) was addedto this mixture. The mixture was stirred and separated into an aqueouslayer and a solvent layer by centrifugation at 10,000 rpm for 3 min. Theaqueous layer was separated, and onto this aqueous layer was gentlylayered a 2-fold amount of ethanol. The content was slowly stirred witha glass rod to allow the DNA to wind around the rod.

This DNA was dissolved in 10 mM Tris-HCl solution (pH 8.0, hereinafterabbreviated as TE) containing 1 mM EDTA. This solution was treated withan equivalent amount of chloroform•phenol solution. The aqueous layerwas separated by centrifugation, and a 2-fold amount of ethanol wasadded. The DNA was separated again by the method described above anddissolved in 2 ml of TE.

Reference Example 2

Preparation of DNA fragment containing a gene encoding creatinineamidinohydrolase and recombinant vector containing said DNA fragment

The DNA (20 μg) obtained in Reference Example 1 was partially cleavedwith restriction enzyme Sau3AI (Toyo Boseki Kabushiki Kaisha) and 2-10kbp fragments were recovered by sucrose density gradient centrifugation.Meanwhile, pBluescript KS(+) cleaved with restriction enzyme BamHI (ToyoBoseki Kabushiki Kaisha) was dephosphorylated with bacterial alkalinephosphatase (Toyo Boseki Kabushiki Kaisha). Then, the both DNAs weretreated with T4DNA ligase (1 unit, Toyo Boseki Kabushiki Kaisha) at 16°C. for 12 hr to ligate the DNA. Escherichia coli JM109 competent cell(Toyo Boseki Kabushiki Kaisha) was transformed with the ligated DNA andplated onto a creatine amidinohydrolase activity detection agar medium[0.5% yeast extract, 0.2% meat extract, 0.5% polypeptone, 0.1% NaCl,0.1% KH₂PO₄, 0.05% MgSO₄/7H₂O, 1.15% creatine, 10 U/ml sarcosine oxidase(Toyo Boseki Kabushiki Kaisha), 0.5 U/ml peroxidase (Toyo BosekiKabushiki Kaisha), 0.01% o-dianisidine, 50 μg/ml ampicillin and 1.5%agar]. The activity of creatine amidinohydrolase was detected using, asthe indices, the colonies grown in the above-mentioned medium andstained in brown. The colonies (ca. 1×10⁵) of the transformant wereobtained per DNA 1 μg used.

About 12,000 colonies were screened, and 6 colonies were found stainedin brown. These strains were cultured in LB liquid medium (1%polypeptone, 0.5% yeast extract, 0.5% NaCl, 50 μg/ml ampicillin) andcreatine amidinohydrolase activity was determined, as a result of whichcreatine amidinohydrolase activity was detected in every strain. Theplasmid of the strain which showed the highest creatine amidinohydrolaseactivity contained ca. 5 kbp insert DNA fragment, and this plasmid wasnamed pCRH17.

Then, the insert DNA of pCHR17 was cleaved with restriction enzymesEcoRV (Toyo Boseki Kabushiki Kaisha) and Pst1 (Toyo Boseki KabushikiKaisha), and ligated to pBluescript KS(+) cleaved with said restrictionenzymes to prepare pCRH173.

Example 1

Preparation of recombinant plasmid pCRH273 by mutating creatineamidinohydrolase gene

The region of from β-galactosidase structural gene derived from thevector to the upstream region of the creatine amidinohydrolasestructural gene of the insert DNA was deleted from the recombinantplasmid pCRH173 of Reference Example 2, using the synthetic DNA depictedin SEQ ID No:3 and a commercially available mutation introduction kit(Transformer™; Clonetech) to prepare recombinant plasmid pCRH173M. Thedetailed method for introducing the mutation was given in the protocolattached to the kit.

The pCRH173M was cleaved with restriction enzyme EcoRI (Toyo BosekiKabushiki Kaisha) and self-ligated to prepare pCRH273 (FIG. 1).

Example 2

Selection of candidate cell lines producing the objective mutantcreatine amidinohydrolase

A commercially available Escherichia coli competent cell (E. coliXLI-Red; Clonetech) was transformed with the pCRH273 prepared in Example1, and the entire amount thereof was inoculated to 3 ml of LB liquidmedium (1% polypeptone, 0.5% yeast extract, 1.0% NaCl) containingampicillin (50 μg/ml; Nakarai Tesque), which was followed by shakeculture overnight at 37° C. A plasmid was recovered from the entireamount of this culture by a conventional method. The commerciallyavailable Escherichia coli competent cell (E. coli JM109, Toyo BosekiKabushiki Kaisha) was transformed again with this plasmid and platedonto a creatine amidinohydrolase activity detection agar medium, whichwas then incubated overnight at 37° C. The cell lines which showed astrong expression of the creatine amidinohydrolase activity, i.e., thestrains which showed a deep color development, were selected from themutant creatine amidinohydrolase library thus obtained.

Example 3

Screening of creatine amidinohydrolase-producing cell line having areduced Km value

The candidate cell lines selected in Example 2 were inoculated to 3 mlof TB medium (1.2% polypeptone, 2.4% yeast extract, 0.4% glycerol,0.0231% KH₂PO₄, 0.1254% K₂HPO₄) containing ampicillin (200 μg/ml) andshake-cultured overnight at 37° C. The cells were recovered from 1 ml ofthe culture by centrifugation, and a crude enzyme solution was preparedtherefrom by rupture with glass beads. Using the crude enzyme solutionthus obtained and following the above-mentioned activity determinationmethod, creatine amidinohydrolase was determined. Meanwhile, using anactivity determination reagent having a 1/10 substrate concentration,the creatine amidinohydrolase activity was determined in the samemanner. The cell line wherein the ratio of the two kinds of the activitymeasures (activity with 1/10 substrate concentration+activity obtainedby conventional manner) increased beyond that of a wild creatineamidinohydrolase was selected as a mutant having a reduced Km value.

About 20,000 cell lines were screened by the above method, and threemutant cell lines having a smaller Km value for creatine were obtained,and the respective recombinant plasmids thereof were named pCRH273M1(FERM BP-5374), pCRH273M2 (FERM BP-5375) and pCRH273M3 (FERM BP-5376).

Example 4

Preparation of creatine amidinohydrolase from Escherichia coli JM109(pCRH273M1)

TB medium (6 L) was dispensed to 10 L jar fermentors, and subjected toautoclaving at 121° C. for 15 min. After allowing them to cool, 50 mg/mlampicillin (Nakarai Tesque) and 200 mM IPTG (Nippon Seika Corp.), whichhad been separately sterilized by filtration, were added by 6 ml each.To this medium was added 60 ml of the culture of Escherichia coli JM109(pCRH273M1)(FERM BP-5374) after previous shake culture at 30° C. for 24hr, which was followed by aeration culture at 37° C. for 24 hr. Theactivity of creatine amidinohydrolase after the completion of theculture was 8.7 U/ml.

The above-mentioned cells were collected by centrifugation, andsuspended in 50 mM phosphate buffer, pH 7.0.

The cells in this suspension were ruptured with a French press andsubjected to centrifugation to give a supernatant. The obtained crudeenzyme solution was subjected to ammonium sulfate fractionation,desalting with Sephadex G-25 (Pharmacia Biotech) gel filtration andpurified by octyl Sepharose CL-6B (Pharmacia Biotech) columnchromatography to give a purified enzyme product. The standard creatineamidinohydrolase product obtained by this method showed a nearly singleband by SDS-PAGE and had a specific activity then of 18.4 U/mg protein.

Table 1 shows the purification performed so far. Table 2 showsphysicochemical properties of the creatine amidinohydrolase obtained bythe above methods.

TABLE 1 Purification of Cratine amidinohydrolase from Escherichia coliJM109 (pCRH273M1) Total Activity Specific activity Yield Step (U)(U/mg-protein) (%) French press rupture 52200 100.0 (NH₄)₂SO₄precipitation - redissolution 49746 8.3 95.3 Sephadex G-25 46927 10.389.9 Octyl Sepharose CL-6B 33094 18.4 63.4

TABLE 2 Physiochemical properties of creation amidinohydrolase purifiedfrom Escherichia coli JM109 (pCRH273M1) Item Physicochemical propertiesAction creatine + H₂O → sarcosine + urea Optimal temperature ca. 40°C.-50° C. Optimal pH ca. 8.0-9.0 Thermal stability ca. 50° C. (50 mMpotassium phosphate buffer, pH 7.5, 30 min treatment) pH stability ca.5-8 (40° C., 18 hr preservation) Km value ca. 6.5 mM (creatine)Molecular weight ca. 43,000 (SDS-PAGE) Isoelectric point ca. [3.5] 4.5(Isoelectric focusing)

Example 5

Preparation of creatine amidinohydrolase from Escherichia coli JM109(pCRH273M2)

TB medium (6 L) was dispensed to 10 L jar fermentors, and subjected toautoclaving at 121° C. for 15 min. After allowing them to cool, 50 mg/mlampicillin (Nakarai Tesque) and 200 mM IPTG (Nippon Seika Corp.), whichhad been separately sterilized by filtration, were added by 6 ml each.To this medium was added 60 ml of the culture of Escherichia coli JM109(pCRH273M2)(FERM BP-5375) after previous shake culture at 30° C. for 24hr, which was followed by aeration culture at 37° C. for 24 hr. Theactivity of creatine amidinohydrolase after the completion of theculture was 5.6 U/ml.

The above-mentioned cells were collected by centrifugation, andsuspended in 50 mM phosphate buffer, pH 7.0.

The cells in this suspension were ruptured with a French press andsubjected to centrifugation to give a supernatant. The obtained crudeenzyme solution was subjected to ammonium sulfate fractionation,desalting with Sephadex G-25 (Pharmacia Biotech) gel filtration andpurified by octyl Sepharose CL-6B (Pharmacia Biotech) columnchromatography to give a purified enzyme product. The standard creatineamidinohydrolase product obtained by this method showed a nearly singleband by SDS-PAGE and had a specific activity then of 14.3 U/mg protein.

Table 3 shows the purification performed so far. Table 4 showsphysicochemical properties of the creatine amidinohydrolase obtained bythe above methods.

TABLE 3 Purification of creatine amidinohydrolase from Escherichia coliJM109 (pCRH273M2) Total activity Specific activity Yield Step (U)(U/mg-protein) (%) French press rupture 33600 100.0 (NH₄)₂SO₄precipitation - redissolution 25636 7.2 76.3 Sephadex G-25 24326 9.872.4 Octyl Sepharose CL-6B 19689 14.3 58.6

TABLE 4 Phsicochemical properties of ceatine amidinohydralase purifiedfrom Escherichia coli JM109 (pCRH273M2) Item Physicochemical propertiesAction creatine + H₂O → sarcosine + urea Optimal temperature ca. 45°C.-50° C. Optimal pH ca. 8.0-9.0 Thermal stability ca. 40° C. (50 mMpotassium phosphate buffer, pH 7.5, 30 min treatment) pH stability ca.5-8 (40° C., 18 hr preservation) Km value ca. 4.5 mM (creatine)Molecular weight ca. 43,000 (SDS-PAGE) Isoelectric point ca. [3.5] 4.5(isoelectric focusing)

Example 6

Preparation of creatine amidinohydrolase from Escherichia coli JM109(pCRH273M3)

TB medium (6 L) was dispensed to 10 L jar fermentors, and subjected toautoclaving at 121° C. for 15 min. After allowing them to cool, 50 mg/mlampicillin (Nakarai Tesque) and 200 mM IPTG (Nippon Seika Corp.) whichhad been separately sterilized by filtration were added by 6 ml each. Tothis medium was added 60 ml of culture of Escherichia coli JM109(pCRH273M3)(FERM BP-5376) after previous shake culture at 30° C. for 24hr, which was followed by aeration culture at 37° C. for 24 hr. Theactivity of creatine amidinohydrolase after the completion of theculture was 8.3 U/ml.

The above-mentioned cells were collected by centrifugation, andsuspended in 50 mM phosphate buffer, pH 7.0.

The cells in this suspension were ruptured with a French press andsubjected to centrifugation to give a supernatant. The obtained crudeenzyme solution was subjected to ammonium sulfate fractionation,desalting by Sephadex G-25 (Pharmacia Biotech) gel filtration andpurified by octyl Sepharose CL-6B (Pharmacia Biotech) columnchromatography to give a purified enzyme product. The standard creatineamidinohydrolase product obtained by this method showed a nearly singleband by SDS-PAGE and had a specific activity then of 14.8 U/mg protein.

Table 5 shows the purification performed so far. Table 6 showsphysicochemical properties of the creatine amidinohydrolase obtained bythe above methods.

TABLE 5 Purification of creatine amidinohydrolase from Escherichia coliJM109 (pCRH273M3) Total activity Specific activity Yield Step (U)(U/mg-protein) (%) French press rupture 49800 100.0 (NH₄)₂SO₄precipitation - redissolution 43027 8.3 86.4 Sephadex G-25 39989 9.980.3 Octyl Sepharose CL-6B 32021 14.8 64.3

TABLE 6 Phsicochemical properties of ceatine amidinohydralase purifiedfrom Escherichia coli JM109 (pCRH273M3) Item Physicochemical propertiesAction creatine + H₂O → sarcosine + urea Optimal temperature ca. 40°C.-45° C. Optimal pH ca. 8.0-9.0 Thermal stability ca. 40° C. (50 mMpotassium phosphate buffer, pH 7.5, 30 min treatment) pH stability ca.5-8 (40° C., 18 hr preservation) Km value ca. 9.0 mM (creatine)Molecular weight ca. 43,000 (SDS-PAGE) Isoelectric point ca. [3.5] 4.5(isoelectric focusing)

The following Table 7 summarizes the Km values for creatine of the novelcreatine amidinohydrolases of the present invention and wild creatineamidinohydrolase. As is evident from Table 7, the novel creatineamidinohydrolases of the present invention had reduced Km values ascompared to the wild creatine amidinohydrolase.

TABLE 7 Enzyme Km value wild 15.2 mM  pCRH273M1 6.5 mM pCRH273M2 4.5 mMpCRH273M3 9.0 mM

Example 7

Using the purified creatine amidinohydrolase prepared in Example 5 andwild creatine amidinohydrolase, a creatinine determination reagenthaving the following composition was prepared, and the amounts of thecreatine amidinohydrolase necessary for giving a creatininedetermination reagent was compared.

creatine amidinohydrolase of Example 5 20, 40, 60 U/ml or wild cratineamidinohydrolase creatinine amidohydrolase 150 U/ml sarcosine oxidase 7U/ml peroxidase 3 PU/ml MOPS buffer 0.1 M, pH 8.0 Triton X-100 0.1%4-aminoantipyrine 0.15 mM TOOS (aniline derivative) 0.2 mM

The above-mentioned solution (3 ml) was added to a sample (60 μl)containing creatinine (100 mg/dl) and changes in absorbance weredetermined at 37° C. at wavelength 546 nm. The time course results areshown in FIG. 2. In the Figure, “Wild” shows a wild creatineamidinohydrolase and “pCRH273M2” is the creatine amidinohydrolase of thepresent invention.

As is evident from FIG. 2, when the determination was ended in 5minutes, the creatine amidinohydrolase of the present invention enableddetermination with less enzyme amount (ca. ⅓ amount) as compared to thewild creatine amidinohydrolase. It was also confirmed that thereactivity during the latter half of the determination, i.e., when thecreatine in the sample decreased, was fine.

1. A creatine amidinohydrolase having the following physicochemicalproperties: Action: catalyzing the following reaction;creatine+H₂O→sarcosine+urea Optimum temperature: about 40-50° C. OptimumpH: pH about 8.0-9.0 K_(m) value for creatine in a coupling assay usinga sarcosine oxidase and a peroxidase: 3.5-10.0 mM Molecular weight:about 43,000 (SDS-PAGE) Isoelectric point: about 3.5.
 2. A creatineamidinohydrolase having the following physicochemical properties:Action: catalyzing the following reaction; creatine+H₂O→sarcosine+ureaOptimum temperature: about 40-50° C. Optimum pH: pH about 8.0-9.0 K_(m)value for creatine in a coupling assay using a sarcosine oxidase and aperoxidase: 4.5±1.0 mM Molecular weight: about 43,000 (SDS-PAGE)Isoelectric point: about 3.5.
 3. The creatine amidinohydrolase of claim2, which is obtained from Escherchia coli JM109 (pCRH273M2) (FERMBP-5375).
 4. A creatine amidinohydrolase having the followingphysicochemical properties: Action: catalyzing the following reaction;creatine+H₂O→sarcosine+urea Optimum temperature: about 40-50° C. OptimumpH: pH about 8.0-9.0 K_(m) value for creatine in a coupling assay usinga sarcosine oxidase and a peroxidase: 6.5±1.0 mM Molecular weight: about43,000 (SDS-PAGE) Isoelectric point: about 3.5.
 5. The creatineamidinohydrolase of claim 4, which is obtained from Escherchia coliJM109 (pCRH273M1) (FERM BP-5374).
 6. A creatine amidinohydrolase havingthe following physicochemical properties: Action: catalyzing thefollowing reaction; creatine+H₂O→sarcosine+urea Optimum temperature:about 40-50° C. Optimum pH: pH about 8.0-9.0 K_(m) value for creatine ina coupling assay using a sarcosine oxidase and a peroxidase: 9.0±1.0 mMMolecular weight: about 43,000 (SDS-PAGE) Isoelectric point: about 3.5.7. The creatine amidinohydrolase of claim 6, which is obtained fromEscherchia coli JM109 (pCRH273M3) (FERM BP-5376).
 8. A method forproducing the creatine amidinohydrolase of claim 1, comprising culturinga microorganism producing said creatine amidinohydrolase in a nutrientmedium and recovering said creatine amidinohydrolase from the resultingculture.
 9. The method of claim 8, wherein said microorganism isselected from the group consisting of Escherichia coli JM109 (pCRH273M1)(FERM BP-5374), Escherichia coli JM109 (pCRH273M2) (FERM BP-5375) andEscherichia coli JM109 (pCRH273M3) (FERM BP-5376).
 10. A reagent fordetermination of creatine in a sample, comprising the creatineamidinohydrolase of claim 1, a sarcosine oxidase and a composition forthe detection of hydrogen peroxide.
 11. The reagent of claim 10, inwhich the composition for the detection of hydrogen peroxide comprisesan enzyme having a peroxidase activity, a chromophore and a buffer. 12.The reagent of claim 11, in which the enzyme having the peroxidaseactivity is selected from the group consisting of peroxidase,haloperoxidase, bromoperoxidase, lactoperoxidase and myeloperoxidase.13. The reaction of claim 11, in which the chromophore comprises ahydrogen receptor and a coupler.
 14. The reagent of claim 13, in whichthe hydrogen receptor is 4-aminoantipyrine or a3-methyl-2-benzothiazoline-hydrazine derivative.
 15. The reagent ofclaim 13, in which the coupler is an aniline derivative or a phenolderivative.
 16. A method for determining creatine in a sample, whichcomprises measuring the absorbance of the pigment produced by thereaction of the reagent of claim 10 with the sample.
 17. A reagent fordetermination of creatinine in a sample, comprising a creatinineamidohydrolase, the creatine amidinohydrolase of claim 1, a sarcosineoxidase and a composition for the detection of hydrogen peroxide. 18.The reagent of claim 17, in which the composition for the detection ofhydrogen peroxide comprises an enzyme having a peroxidase activity, achromophore and a buffer.
 19. The reagent of claim 18, in which theenzyme having the peroxidase activity is selected from the groupconsisting of peroxidase, haloperoxidase, bromoperoxidase,lactoperoxidase and myeloperoxidase.
 20. The reagent of claim 18, inwhich the chromophore comprises a hydrogen receptor and a coupler. 21.The reagent of claim 20, in which the hydrogen receptor is4-aminoantipyrine or a 3-methyl-2-benzothiazoline-hydrazine derivative.22. The reagent of claim 20, in which the coupler is an anilinederivative or a phenol derivative.
 23. A method for determiningcreatinine in a sample, which comprises measuring the absorbance of thepigment produced by the reaction of the reagent of claim 17 with thesample.
 24. A method of preparing a creatine amidinohydrolasecomprising: (i) mutating (a) the nucleic acid sequence of SEQ ID NO:2 or(b) a nucleic acid sequence encoding the amino acid sequence of SEQ IDNO:1 to provide mutant nucleic acid sequences, (ii) determining Kmvalues for creatine of proteins encoded by the mutant nucleic acidsequences in a coupling assay using a sarcosine oxidase and aperoxidase, (iii) selecting and isolating a desired mutant nucleic acidsequence that encodes a creatine amidinohydrolase having the followingphysicochemical properties: Action: catalyzing the following reaction:creatine+H ₂ O→sarcosine+urea Km values for creatine in a coupling assayusing a sarcosine oxidase and a peroxidase: 3.5-10.0 mM, Molecularweight: about 43,000 (SDS-PAGE) Isoelectric point: about 4.5 Optimumtemperature: about 40-50° C. (at pH of about 7.5 ) Optimum pH: about8.0-9.0 (at a temperature of about 37° C.) (iv) expressing the desiredmutant nucleic acid sequence in a host to produce creatineamidinohydrolase, and (v) harvesting the produced creatineamidinohydrolase.
 25. The method of claim 24, wherein the sarcosineoxidase is originated from the genus Arthrobacter, Corynebacterium,Alcaligenes, Pseudomonas, Micrococcus, or Bacillus.
 26. A method ofpreparing a creatine amidinohydrolase comprising: (i) selecting (a) anucleic acid sequence of SEQ ID NO:2 or (b) a nucleic acid sequenceencoding the amino acid sequence of SEQ ID NO:1 to provide a sourcenucleic acid sequence, (ii) mutating the source nucleic acid sequence toprovide mutant nucleic acid sequences that encode mutant creatineamidinohydrolases, (iii) selecting a mutant nucleic acid sequence thatencodes a creatine amidinohydrolase which has a reduced Km value ascompared to the Km value of creatine amidinohydrolase encoded by thesource nucleic acid sequence by: (A) determining a first activity ofcreatine amidinohydrolase encoded by the source nucleic acid sequencewith a first concentration of creatine and a second activity of creatineamidinohydrolase encoded by the source nucleic acid sequence with asecond concentration of creatine, wherein the second concentration ofcreatine is less than the first concentration of creatine, (B)determining a first activity of the mutant creatine amidinohydrolasewith the first concentration of creatine and a second activity of themutant creatine amidinohydrolase with the second concentration ofcreatine, wherein the second concentration of creatine is less than thefirst concentration of creatine, (C) calculating a ratio of the secondactivity of the creatine amidinohydrolase encoded by the source nucleicacid sequence divided by the first activity of the creatineamidinohydrolase encoded by the source nucleic acid sequence, (D)calculating a ratio of the second activity of the mutant creatineamidinohydrolase divided by the first activity of the mutant creatineamidinohydrolase, (E) comparing the ratio calculated in step (iii)(C) tothe ratio calculated in step (iii)(D), wherein a mutant creatineamidinohydrolase that has a reduced Km value as compared to the Km valueof creatine amidinohydrolase encoded by the source nucleic acid sequencehas a greater ratio than the ratio for creatine amidinohydrolase encodedby the source nucleic acid, (iv) selecting and isolating a desiredmutant nucleic acid sequence that encodes a creatine amidinohydrolasehaving the following physicochemical properties: Action: catalyzing thefollowing reaction: creatine+H ₂ O→sarcosine+urea Km values for creatinein a coupling assay using a sarcosine oxidase and a peroxidase: 3.5-10.0mM, Molecular weight: about 43,000 (SDS-PAGE) Isoelectric point: about4.5 Optimum temperature: about 40-50° C. (at pH of about 7.5 ) OptimumpH: about 8.0-9.0 (at a temperature of about 37° C.) (v) expressing thedesired mutant nucleic acid sequence in a host to produce creatineamidinohydrolase; and (vi) harvesting the produced creatineamidinohydrolase.